Diversity of Pectobacteriaceae Species in Potato Growing Regions in Northern Morocco

microorganisms Article Diversity of Pectobacteriaceae Species in Potato Growing Regions in Northern Morocco Saïd Oulghazi 1,2, Mohieddine Moumni 1, Slimane Khayi 3 ,Kévin Robic 2,4, Sohaib Sarfraz 5, Céline Lopez-Roques 6,Céline Vandecasteele 6 and Denis Faure 2,* 1 Department of Biology, Faculty of Sciences, Moulay Ismaïl University, 50000 Meknes, Morocco; [email protected] (S.O.); [email protected] (M.M.) 2 Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 91198 Gif-sur-Yvette, France; [email protected] 3 Biotechnology Research Unit, CRRA-Rabat, National Institut for Agricultural Research (INRA), 10101 Rabat, Morocco; [email protected] 4 National Federation of Seed Potato Growers (FN3PT-RD3PT), 75008 Paris, France 5 Department of Plant Pathology, University of Agriculture Faisalabad Sub-Campus Depalpur, 38000 Okara, Pakistan; [email protected] 6 INRA, US 1426, GeT-PlaGe, Genotoul, 31320 Castanet-Tolosan, France; [email protected] (C.L.-R.); [email protected] (C.V.) * Correspondence: [email protected] Received: 28 April 2020; Accepted: 9 June 2020; Published: 13 June 2020 Abstract: Dickeya and Pectobacterium pathogens are causative agents of several diseases that affect many crops worldwide. This work investigated the species diversity of these pathogens in Morocco, where Dickeya pathogens have only been isolated from potato fields recently. To this end, samplings were conducted in three major potato growing areas over a three-year period (2015–2017). Pathogens were characterized by sequence determination of both the gapA gene marker and genomes using Illumina and Oxford Nanopore technologies. We isolated 119 pathogens belonging to P. versatile (19%), P. carotovorum (3%), P. polaris (5%), P. brasiliense (56%) and D. dianthicola (17%). Their taxonomic assignation was confirmed by draft genome analyses of 10 representative strains of the collected species. D. dianthicola were isolated from a unique area where a wide species diversity of pectinolytic pathogens was observed. In tuber rotting assays, D. dianthicola isolates were more aggressive than Pectobacterium isolates. The complete genome sequence of D. dianthicola LAR.16.03.LID was obtained and compared with other D. dianthicola genomes from public databases. Overall, this study highlighted the ecological context from which some Dickeya and Pectobacterium species emerged in Morocco, and reported the first complete genome of a D. dianthicola strain isolated in Morocco that will be suitable for further epidemiological studies. Keywords: Pectobacterium; Dickeya; plant pathogen; potato tuber; genome; field sampling 1. Introduction Pectinolytic Pectobacterium and Dickeya spp. are causative agents of severe diseases in a wide range of plants of high economic value [1,2]. On potato tubers and stems, the diseases caused by pectinolytic pathogens are soft rot and blackleg, respectively. These pathogens produce a large set of extracellular enzymes that degrade the plant cell wall, resulting in plant tissue decay and maceration. This rotting process causes losses in the production of potato tubers sold both on the food market and as certified seed tubers [3]. The pathogens may be acquired by the host plants from soil and/or from contaminated seed tubers [4]. On plants, pathogen populations remain at a low level in asymptomatic Microorganisms 2020, 8, 895; doi:10.3390/microorganisms8060895 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 895 2 of 16 plant tissues, and may become particularly destructive when environmental conditions favor their proliferation and the expression of virulence factors. P. atrosepticum was considered as the primary pathogen responsible for the rotting of stored potato tubers and wilting of potato plants under temperate climates [4]. Other Pectobacterium species frequently associated with damage of potato crops are P. carotovorum, P. brasiliense, P. parmentieri and P.polaris [5–9]. Some Pectobacterium species have also been characterized in some specific areas, such as P. peruviense strains isolated from tubers in Peru, and P. punjabense species from symptomatic potato plants in Pakistan [10,11]. P. odoriferum exhibits a very wide host range, including potato plants [12], while some other species were characterized by a more restricted host range, at least in the fields. Thus, P. wasabiae was isolated from symptomatic Japanese horseradish [13]; P. betavasculorum was reported almost exclusively on sugar beet [14]; P. aroidearum exhibits a preference for some monocotyledonous plants [15]; P. zantedeschiae strains were isolated from Zantedeschia spp. (Calla lily) [16]; and P. actinidiae from symptomatic Actinidia chinensis (kiwi fruit) [17]. Recently, some other species, isolated from surface waters, have also been described: P. fontis, P. aquaticum and P. versatile [7,18,19]. Altogether, 16 Pectobacterium species have been described so far [7]. A limited number of Dickeya species, i.e., D. dianthicola, D. dadantii and D. solani, have been associated with symptoms on potatoes. D. dianthicola was first reported on potatoes in the Netherlands in the 1970s, and has been detected since then in many other European countries [20]. D. dadantii causes soft rot disease in several members of the Solanaceae family, including the potato [21]. Another virulent species, namely D. solani, spread rapidly throughout Western Europe [22] and in Russia [23], and into other countries such as Turkey [24], Georgia [25] and Brazil [26]. During the past decade, the taxonomy of Dickeya and Pectobacterium species was revisited following genomic studies bearing on international culture collections and diverse ecosystems around the world [27,28]. By now, the genus Dickeya encompasses 10 species: D. aquatica, D. chrysanthemi, D. dadantii, D. dianthicola, D. fangzhongdai, D. lacustris, D. paradisiaca, D. solani, D. undicola, and D. zeae [22,29–34]. Bacteria belonging to this genus cause plant diseases in temperate, tropical and subtropical climates [35]. The unambiguous identification of Dickeya and Pectobacterium species is crucial for epidemiological purposes, to develop appropriate prophylactic approaches and quality controls in national and international trade exchanges. Multi-Locus Sequence Analysis (MLSA) provides relevant information for a better understanding of speciation, and hence for proposing pertinent taxa delineations [15,36]. MLSA may exploit gene sequences, obtained by PCR-sequencing of several loci or by whole genome sequencing. Among the loci commonly included in MLSA, the rrs sequence is poorly informative at a species level, while the gapA gene appeared as an appropriate marker to discriminate the different Dickeya and Pectobacterium species [10,18,19,31,32,37]. Taxonomy of Dickeya and Pectobacterium gained precision and robustness with additional genome analyses, such as average nucleotide identity (ANI) and in silico DNA–DNA hybridization (isDDH) [38]. Comparative genomics is also used to identify species-specific DNA regions. Analysis of the functions encoded by these DNA regions allows the prediction of species-specific metabolic traits. This knowledge contributes to the understanding of both the taxonomy and ecology of the Dickeya and Pectobacterium pathogens [22,30–32,39]. P. atrosepticum, P. carotovorum and P. brasiliense were described in Morocco as the main causative agents of blackleg and soft rot diseases in potato crop [40–43]. In 2016, D. dianthicola was described for the first time in the North of Morocco [44]. In this respect, the main objectives of this study were: (i) to investigate the species composition of the Moroccan Dickeya and Pectobacterium populations, collected between 2015 and 2017 from diseased potato tubers and stems, (ii) to compare the aggressiveness of some identified pathogens belonging to different species, and (iii) to propose a complete genome of the emerging pathogen D. dianthicola in Morocco, that could be used for further studies as a reference genome. This work represents the most important sampling effort of the Pectobacterium and Dickeya potato pathogens in Morocco over the past decade. Microorganisms 2020, 8, 895 3 of 16 2. Materials and Methods 2.1. Sampling and Isolation of Pectinolytic Bacteria In 2015, 2016 and 2017, blackleg symptoms were searched for in potato fields in four regions (Meknes, Guigo, Boumia and Larache) in Northern Morocco. Pectinolytic bacteria were isolated from symptomatic plant tissues using crystal violet pectate (CVP) medium as described previously [45]. The CVP plates were incubated at 28 ◦C for 3 days and colonies that had formed pits were re-streaked onto Tryptone (5 g/L) yeast extract (3 g/L) agar medium (TY). The purified isolates were spotted again on CVP to confirm the pectinolytic activity. The obtained cultures from single colonies were stored in 25% glycerol at 80 C. − ◦ 2.2. Molecular Characterization of Pectobacterium and Dickeya Isolates The primer couples Y1/Y2 and ADE1/ADE2 (Table S1) were used for the identification of isolates belonging to Pectobacterium and Dickeya genera [46,47]. The reaction was carried out in a final volume of 25 µL, containing 1 µL of bacterial DNA (50 ng/µL), 2.5 µL of PCR buffer (10 ), 2 µL of × MgCl2 (25 mM), 2.5 µL of dNTPs (1 mM), 1U Taq polymerase and 1 µL of each primer (1 µM) and water. The temperature settings for PCR were the same as described before [46,47]. The analysis of PCR products was done by electrophoresis on 2% agarose gels, using PCR products of

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